The NH2-terminal Ig Domains Of Insect Projectin Could Serve As Elastic Elements

Abstract

AbstarctThe connecting C-filaments of insect indirect flight muscles have been proposed as one of the elements providing muscle elasticity for the asynchronous muscle physiology of derived insects. Two large modular proteins, kettin/Sallimus and projectin make up these filaments, and for both proteins the N-terminal sequences span the extensible I-band and are proposed as the elastic segments. The C-filaments have not been studied in insects, such as dragonflies, crickets, and Lepidoptera with muscles which are largely synchronous in physiology and display different levels of muscle stiffness. In this paper we focus our efforts on the projectin protein of several insects with synchronous flight muscles; namely dragonfly, cricket, and moth. We provide evidence for the localization of projectin over the sarcomere I-Z-I region that is consistent with the existence of C-filaments in synchronous flight muscles. Additionally, we determine the sequences for the NH 2 -terminal region of projectin in these insects and describe the presence of alternative splice variants. Using predictors of intrinsically disordered regions, we identify possible unfolded segments, especially around the short linker sequences found between the NH 2 Ig domains. We propose a possible picture of projectin NH 2 -terminal region organized as different segments contributing elastic responses to stretch by either unfolding of highly disordered sequences (PEVK) or reorientation of domains by bending or twisting of disordered linkers between the Ig domains.

Elastic Proteins in the Flight Muscle of Manduca Sexta

“…The predicted sizes for these isoforms are consistent with the bands identified as projectin by Western blot analysis and confirm that neither the 1.6 nor 2.0 MDa can be projectin isoforms. This is also supported by the molecular analysis of projectin in B. mori and D. plexippus [22,40]. Rotary shadowing of a 600 kDa projectin isoform from locust showed an elongated molecule measuring 260nm in length [45].…”

“…All splice sites were verified by either RNA-sequencing data or RT-PCR sequences. The analyses of the NH 2 -terminus and PEVK sequences of M. sexta projectin have been described elsewhere together with the complete gene structure for the Monarch butterfly (Danaus plexippus [22,40]). The overall domain organization of projectin is identical to all the projectin proteins characterized so far [15,20,22].…”

Localization of the Elastic Proteins in the Flight Muscle of Manduca sexta

“…Antibodies used are described in Table 1 (see Methods). The NT2 antibody was used to label the N-terminal Ig9 and Ig10 peptides of projectin [ 27 ]. The results showed that NT2 signals was co-localized with α-actinin ( Figure 4 A) which indicate that, like kettin in Drosophila and Lethocerus flight muscle [ 28 ] and titin in vertebrate muscle [ 29 , 30 ], the N-terminus of projectin in DLM 1 appears to be anchored to the Z-band.…”

“…The results showed that at short sarcomere length (3.1 μm), the two stripes were close together, and the two stripes gradually move away from each other as the myofibril is stretched to longer sarcomere length. The position of the 3B11 epitope is located at the C-terminus of the projectin molecule [ 27 ]. While the 3B11 fluorescent signals were also observed next to the Z-band, they were further away from the Z-band compared to the MAC150 signals at slack length ( Figure 4 A).…”

Myofilament proteins in the synchronous flight muscles of Manduca sexta show both similarities and differences to Drosophila melanogaster

“…The gene structure, isoforms and expression patterns for these two genes is described elsewhere (Ayme- Southgate et al, 2013Southgate et al, , 2014Yuan et al, in press). …”